Ontogenic development of cardiomyocytes derived from transgene-free human induced pluripotent stem cells and its homology with human heart

Life Sciences 92 (2013) 63–71

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Ontogenic development of cardiomyocytes derived from transgene-free human induced pluripotent stem cells and its homology with human heart Glen Lester Sequiera, Ashish Mehta, Ting Huay Ooi, Winston Shim ⁎ Research and Development Unit, National Heart Centre Singapore, Singapore

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Article history: Received 20 June 2012 Accepted 26 October 2012 Keywords: Induced pluripotent stem cells Cardiomyocytes Differentiation Gene expression

a b s t r a c t Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study, we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs. Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR. Key findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers, generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers, followed by early cardiac committed markers, structural and ion channel genes. Furthermore, our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin, actinin, myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally, microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol, reduced Na + influx with quinidine, prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil. Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiacspecific molecular, structural, and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine. © 2012 Elsevier Inc. All rights reserved.

Introduction Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) serve as an effective in vitro tool for studying cardiac developmental processes (Nelson et al., 2010), drug toxicity screenings (Mehta et al., 2011) and disease modeling to unravel the underlying molecular mechanisms of disease manifestations (Carvajal-Vergara et al., 2010). The application of pluripotent stem cell derived CMs would act as an attractive human in vitro model in which early cardiotoxicity prediction could be performed pre-emptively in the initial phase of lead compound selection, expediting drug discovery process. Moreover, transgene free hiPSC derived-CMs being patient-specific are free from ethical concerns and ⁎ Corresponding author at: National Heart Centre Singapore, 17, Third Hospital Avenue, Mistri Wing, Singapore 168752, Singapore. Tel.: + 65 64350752; fax: + 65 62263972. E-mail address: [email protected] (W. Shim). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.10.020

immunological complications faced by human embryonic stem cell (hESC). Nevertheless, the human heart is an extremely complex organ composed of many distinct functional cell types of cardiomyocytes (nodal, atrial and ventricular) as well as other cell types. This makes the development of a humanized in vitro model to recapitulate the in vivo complexity highly challenging. The hiPSC-CMs are indistinguishable from ESC-derived CMs in sarcomeric organizations, electrophysiological properties and response to cardiac drugs such as β-adrenergic stimulation (Mehta et al., 2011; Zhang et al., 2009). Similar to ESC-derived CMs, iPS-derived CMs resemble fetal myocytes with embryoid body formation mimicked heart development in embryos (Sartiani et al., 2007) whereby an upregulated expression of mesoderm and cardiomesoderm markers in the initial stages was succeeded by an increasing expression of cardiac-specific transcription factors and ultimately the cardiac-specific structural genes (Mehta et al., 2011; Zwi et al., 2009). Electrophysiological studies revealed the presence of atrial-like, ventricular-like and nodal-like

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subtypes (He et al., 2003). Ultrastructural analysis (Snir et al., 2003) and molecular signatures (Synnergren et al., 2008) and ion channel expression (Asp et al., 2010) revealed their close similarities to those of human heart tissue. However, only a limited number of studies have directly compared human ES-derived CMs to human heart muscle (Beqqali et al., 2006; Cao et al., 2008). Furthermore, there is a lack of published report on ontogenic development of human iPSC-derived CMs and their similarities with human heart. Our present study involved generating viral-free hiPSC through episomal vectors, and delineating developmental ontogeny of the derived cardiomyocytes in comparison to human adult heart tissues and validating their pharmacological responses to common cardiac active agents.

Immunostaining

Materials and methods

Colonies of iPSC and single cells generated from beating clusters were seeded on matrigel and gelatin coated glass slides, respectively. Both cell types were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 (Sigma-Alrich, MO, USA) and blocked with 5% bovine serum albumin (Sigma-Alrich, MO, USA). Human iPS colonies were stained for 1 h with primary antibodies targeting pluripotency markers, Oct-4, SSEA4, Tra-1-60 and Tra-1-80 (all Millipore, MA, USA), whereas cardiomyocytes (CMs) were stained with primary antibodies, α-actinin (Sigma-Alrich, MO, USA), MLC (USBiologicals, MA, USA), titin (DHSB, Iowa, USA) and cardiac troponin (USBiologicals, MA, USA). Samples were washed and incubated with respective secondary antibodies (Invitrogen, CA, USA) for 1 h and subsequently counterstained with DAPI. Slides were examined under Zeiss Axlovert 200 M LSM5 PASCAL confocal microscope (Carl Zeiss Inc., USA).

Reprogramming, cell lines and culture

Quantitative and semi-quantitative PCR

Human dermal fibroblasts (Lonza, Basel, Switzerland) were cultured in DMEM (Invitrogen, CA, USA) with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, UT), 0.1 mM non-essential amino acids (Invitrogen, CA, USA), 2.0 mM Glutamax (Invitrogen, CA, USA), and 0.1 mM ß-mercaptoethanol (Invitrogen, CA, USA). Fibroblasts were passaged every 4–5 days with TrypLE (Invitrogen, CA, USA) and seeded in new flasks at a density of 15,000–20,000 cell/cm 2. Human dermal fibroblasts were co-transfected with two oriP/EBNA1-based episomal plasmids (pEP4 EO2S CK2M EN2L, pEP4 EO2S ET2K from Addgene Inc., MA, USA) via nucleofection (NHDF — VPD-1001 with U-20 program, Amaxa, Walkersville, MD) and the transfected fibroblasts (~1.0 × 10 6 cells per nucleofection) were directly plated to 10-cm mitomycin-C inactivated MEF-seeded dishes as reported previously (Mehta et al., 2011). Post-day 4 nucleofection, fibroblast growth medium was replaced with hESC medium (Mehta et al., 2008, 2011) with 100 ng/mL bFGF (R & D Systems, USA) and these plates were cultured for 3–4 weeks. Medium was replaced every 2–3 days with fresh medium and plates were observed for sign for cell aggregation regularly. Small aggregates/colonies with morphology similar to hiPS colonies were readily visible between day 35 and 40 post-transfection. These individual colonies were picked manually from the parent plate and plated on 1% matrigel coated dishes and maintained in mTeSR1 medium (StemCell Technologies, VA, Canada) for further culturing and experiments. Human iPSC lines were maintained on 1% matrigel (BD Biosciences, CA, USA) and grown in chemically defined mTeSR1 medium (StemCell Technologies, VA, Canada). Care was taken to manually remove the differentiated areas (b 10%) with the help of a scraper before passaging. Cells were washed with DMEM-F12 (Invitrogen, USA) and incubated with 1 mg/ml dispase (Sigma, USA) for 5–7 min. Cells were then washed with DMEM-F12 and colonies scrapped from the culture dishes. Controlled tituration of the colonies generated small clumps for replating on new matrigel coated dishes. Cells were fed daily with fresh mTeSR1 medium and passaged every 6–7 days and maintained at 37 °C under 5% CO2 in air at 95% humidity.

For reverse-transcription polymerase chain reaction (RT-PCR) analysis, adult human heart RNA (Clontech, USA), undifferentiated hiPS/hES cells (day 0) and differentiating EBs at different time points were utilized. RNA was isolated with the RNeasy kit (Qiagen GmbH, Hilden, Germany). One μg of total RNA was converted to complementary DNA by Superscript II first-strand synthesis system (Invitrogen, CA, USA). cDNA template (5 ng) was used from each sample and SYBR green real-time PCR studies were performed using Quantifast kit (Qiagen GmbH, Hilden, Germany) and primer (Supplementary Table 1) as per the kit instructions. Samples were cycled with RotorGene Q (Qiagen GmbH, Hilden, Germany) as follows: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s extension at 60 °C. All experiments were performed in triplicates. Relative quantification was calculated according to the ΔΔCt method for quantitative realtime PCR (using an endogenous control gene, GAPDH). For each gene, the expression at a specific day was then normalized by its baseline values. For semi-quantitative RT-PCR 5 ng cDNA template was used for each sample and PCR was performed for 30 cycles 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s, with initial deactivation at 95 °C for 5 min and final extension at 72 °C for 7 min in GeneAmp PCR system 2700 (Applied Biosystems, USA). PCR products were electrophoresed on 1.5% agarose gel with ethidium bromide (Sigma-Alrich, MO, USA) and bands were visualized and recorded using Geldoc XR (Bio-Rad, USA). List of primers used in the study are listed in Supplementary Table 1.

Embryoid body formation and cardiomyocyte differentiation Pluripotent stem cell colonies were dispersed into small clumps with dispase (1 mg/ml) and placed in low adhesion culture dishes in EB medium (Mehta et al., 2010) along with or without 5 μM of SB203580 (Calbiochem, USA) for 8 days (Mehta et al., 2011). Subsequently, EBs were plated on 0.1% gelatin coated dishes in EB media without SB203580. Beating areas were typically observed around day 11 − 14 from EB formation. Beating areas were manually cut after day 21 of differentiation and utilized for various experiments.

Teratoma generation All animal experiments were conducted following experimental protocols approved by the SingHealth Institutional Animal Care and Use Committee, in full compliance with Singapore laws and regulations, and followed the guidelines of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Severe combined immunodeficient (SCID) mice, 6 weeks old, weighing 20–23 g, were obtained from SingHealth Experimental Medicine Centre, and were anesthetized with isoflurane and approximately 1 × 106 hiPSCs were injected into the kidney capsule as previously described (Ritner and Bernstein, 2010). Mice were monitored regularly for teratoma formation and then were killed by asphyxiation at 7 weeks to extract the tumors. Teratoma was cleaned from host tissue, washed with PBS saline and fixed and processed for H&E staining. Microelectrode array (MEA) recordings To characterize the electrophysiological properties of the hiPSCMs, a microelectrode array (MEA) recording system (Multichannel

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Systems, Reutlingen, Germany) was used. Contracting areas were micro-dissected and plated on gelatin coated MEA plates. The clusters were allowed to adjust for 72 h before performing any recording. All clusters were monitored for their beating abilities (beats/min) under the microscope during the 72 h period. Clusters that maintained relatively uniform beating rates were then subjected to drugs. The MEA system allows simultaneous recordings from 60 titanium nitridecoated gold electrodes (30 μm) at high spatial (200 μm) and temporal (15 kHz) resolutions. To assess the effects of different drugs on the electrophysiological properties, the stock drugs were diluted in medium (2 mL). MEA clip along with the beating clusters was maintained on 37 °C throughout the duration of experiments. Care was also taken that all buffers including the medium utilized during all experimentation were pre-warmed to 37 °C. The tested drugs include isoproterenol hydrochloride (Iso), quinidine, verapamil and E-4031. All extracellular recordings were performed for 180 s at baseline and at 5 min after drug application at 37 °C. Data was recorded using MC Rack software (Multichannel System) for all drugs. Statistical analysis Comparisons at each time point were conducted using analysis of variance (ANOVA) followed by post-hoc test, and all data are presented as mean values ± S.E.M. Differences were considered statistically significant at p ≤ 0.05.

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neuroepithelial rosettes, (ectoderm; Fig. 1Ea), cartilage (mesoderm; Fig. 1Eb) and secretory tubules (endoderm; Fig. 1Ec). Cardiomyocyte differentiation and characterization Cardiomyocyte differentiation was performed via the EB method. After 8 days in suspension with 5 μM SB203580, EBs were placed on gelatin-coated culture dishes. Rhythmically contracting areas appeared as early as 11–12 days post-differentiation initiation. These contracting clusters continued to beat for several weeks (6–9 weeks as observed) post induction. It was interesting to note that out of the 5 cell lines (MSnviPSdAF1, 3, 4, 5, 7) generated from reprogramming only 2 cell lines (MSnviPSdAF4 and 7) were able to demonstrate consistent contractions with efficiencies ranging from 15 to 25%. The other 3 cell lines gave cardiomyocytes intermittently with low efficiencies of 1–5% (Fig. 2A). Henceforth, all the data demonstrated herein was performed on MSnviPSdAF7 (AC7) and MSnviPSdAF4 (AC4). For confirming the presence of cardiomyocytes, contracting clusters were dissociated and immunostained. Human iPSC-CMs stained positively for the sarcomeric cardiac actinin (Fig. 2B), myosin light chain (Fig. 2C), cardiac troponin (Fig. 2D) and titin (Fig. 2E), confirming their cardiac nature. Interestingly, these positively stained cardiomyocytes demonstrated a less organized striated pattern indicating towards early stages of myocytes. However, Z-bands and A-bands were clearly visible in these cardiomyocytes (Fig. 2B–E, arrows). Ontogenic development of hiPSC-CMs

Results Characterization of human induced pluripotent stem cells Adult human dermal fibroblasts were subjected to nucleofection with episomal vectors with 7 factors (Oct-4, Sox2, Klf4, c-myc, Nanog, Lin28 and SV40L) gave rise to five transformed colonies with morphologies similar to hESCs in 35–40 days. These colonies were hand-picked from the parent plate (10 cm culture dishes) and plated on matrigel coated dishes and maintained in mTeSR1 medium. In order to confirm that reprogramming of fibroblasts was successful, we performed whole colony immunofluorescence staining of these hiPSCs with known pluripotency markers. Results showed strong nuclear staining for Oct-4 (Fig. 1Aa) and surface staining for SSEA4, Tra-1-60 and Tra-1-81 (Fig. 1Ab–d), respectively, providing first evidence of their pluripotency. We next performed semi-quantitative gene expression evaluation of pluripotency markers, Oct4, Sox2 and Nanog (Fig. 1B). PCR results clearly indicated that hiPSCs maintained high levels of the three pluripotency markers (Fig. 1B). Moreover, adult dermal fibroblast cells too expressed low levels of pluripotency markers at gene levels, except Sox2 (Fig. 1B). Since reprogramming of the fibroblasts to generate iPSCs was performed utilizing episomal plasmids, we checked for genetic incorporation of the vector genes with host genome. Genomic PCR data revealed that no genetic incorporation of the vector genes occurred in the iPSC clones (data not shown). Further, karyotypic analysis of these cells demonstrated a normal female phenotype (Supplementary Fig. 1). One of the hallmarks for pluripotent stem cells is their ability to differentiate into three germ layers in vitro as well as in vivo. The hiPSCs were detached and cultivated in suspension to generate 3-dimensional differentiating cell aggregates of embryoid bodies (EBs) (Fig. 1C). Following 10 days of differentiation, EBs were analyzed for bona fide markers of the three germ layers. There was a significant up regulation of markers of ectoderm (Nestin), mesoderm (GATA4) and endoderm (HNF4α), indicating that these iPSCs had the potential to differentiate into all three germ layers (Fig. 1D). Furthermore, transplantation of undifferentiated hiPSC in the kidney capsule of SCID mice generated teratoma in 7 weeks, comprising cell types of

In order to understand the developmental ontogeny of these cardiomyocytes from hiPSC, real-time PCR analysis was performed at different days of differentiation (day 0, 7, 14 and 21). Our results demonstrated a significant decrease (20 folds) in the pluripotency marker, Oct-4 (Fig. 3A) from day 0 to day 7 (relative level on day 0 vs. day 7: 1.0 ± 0.0 vs. 0.039 ± 0.016, n = 3, p b 0.05). These levels were further down regulated, but less significantly as compared to first 7 days of differentiation. A drastic increase in gene expression was observed by day 7 for Isl1, a marker for secondary heart field (Fig. 3A), which subsequently showed a marked decrease by day 14 and 21 (Fig. 3A). We next observed a time-dependent increase in transcriptional factors, GATA4 (mesodermal marker) and NKx2.5, cardiac-committed marker (Fig. 3A) from day 7 to 21. While there was a significant increase in the level of these markers with respect to day 7, these levels were nevertheless markedly lower than adult human heart levels. Finally, we studied gene expression levels of cardiac-specific structural and sarcomeric proteins (cardiac actinin, CAA; cardiac troponin, cTnI; β-myosin heavy chain, MYH7 and myosin light chain 2v, MLC2v). All sarcomeric proteins demonstrated a significant up-regulation of gene expression by day 14 that increased further by day 21 of differentiation (Fig. 3A). Similar results were also observed in cardiac troponin T and ion channel proteins, Cav1.3 encoding the α-1D subunits of the L-type calcium channel and KCNH2 [hERG] mediating the rapid delayed rectifier potassium current (data not shown). Nevertheless, gene expression levels of all sarcomeric proteins as well as ion channel markers were significantly lower when compared to human heart samples (HH) (Fig. 3A). In order to understand the ontogenic development of these cardiomyocytes, we overlaid the normalized expression levels of markers for generating a clearer view of the time frames for upregulation of genes. Our analysis indicated that while Oct-4 downregulation was coupled with Isl1 up-regulation indicating towards mesodermal commitment for cardiogenesis. The down-regulation of Isl1 (day 14) was followed by a significant increase in Nkx2.5 (Fig. 3Ba) and CAA (Fig. 3Bb), suggesting them as the first markers indicative of cardiac commitment of cells. The significant up-regulation of Nkx2.5 (day 14) also correlated increase in important structural proteins, cTnI (Fig. 3Ba) and MLC2v (Fig. 3Bb). Increase in expression

66 G.L. Sequiera et al. / Life Sciences 92 (2013) 63–71 Fig. 1. Characterization of viral free iPSC. A, immunostaining of the undifferentiated hiPSC colonies with Oct-4(a), SSEA-4(b), Tra-1-60(c) and Tra-1-81(d) antibodies followed by counterstaining with DAPI(e-h). Scale bar — 100 μm. B, semi-quantitative gene expression levels of Oct-4(O), Sox-2 (S) and Nanog (N) in four iPSC clones in comparison with hESC line, H9 (positive control) and human foreskin fibroblast (negative control), along with GAPDH (G) as a housekeeping control. Lane 1: H9 hESC, lane 2–5: hiPSC AC1, 3, 4 and 7 respectively, lane 6 — adult fibroblast, lane 7: no template control. C, embryoid bodies (a) generated from clone MSnviPSAF7 show the gene expression of hallmark markers of ectoderm (nestin), mesoderm (GATA4) and endoderm (HNF4α), along with GAPDH in semi-quantitative RT-PCR (b). Lane 1: undifferentiated AC7 hiPSC, lane 2: day 7 EBs of hiPSC-AC7. D, hematoxylin and eosin (H&E) staining of teratoma sections of MSnviPSAF7 showing the presence of ectoderm (neural rosettes), mesoderm (cartilage) and endoderm (secretory tubule). Scale bar — 200 μm.

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Fig. 2. Characterization of iPSC-CM. A, box-plot shows the cardiac differentiation efficiencies of the 5 hiPSC lines. Data represent a mean of 13 independent differentiation experiments. B–E, immunostaining of structural proteins, sarcomeric α-actinin, myosin light chain 2 (MLC2), cardiac troponin-C (cTnC) and titin respectively. Nuclei were counterstained with DAPI in all images. All images have been taken on 63× magnification. Scale bar — 50 μm. Note, arrowhead shows clear immature striation patterns in hiPSC derived CMs.

of these hallmark sarcomeric proteins was coupled with increase in expression of ion channel regulating genes, CACNA1D (Fig. 3Ba) and KCNH2 (Fig. 3Bb). hiPSC-CMs are similar to human heart Our above results, clearly suggest that hiPSC-CMs show expression profiles similar to human cardiac cells. However, to further elaborate if there are similarities in the gene expression levels, we performed correlation analysis between individual genes. These evaluations would rule out possibilities if there were any arbitrary expression observed in the hiPSC-CMs. Our correlation studies indicated that there is a high degree of correlation between expression of several important genes such as, CAA, cTnI, MLC2v, CACNAD1, KCNH2 and KCNQ1 (Table 1), which form the functional early markers of cardiomyocytes. Our results clearly indicate that not just one or two genes were activated, but the whole cardiac transcriptional machinery was active in our differentiation protocol. Interestingly, MYH7 had lower correlation or no correlation with most of the other genes, specifically CAA and CACNAD1 (Table 1).

evaluated effects of calcium L-type channel blocker, verapamil and β-agonist, isoproterenol. Verapamil treatment at 0.1 μM dose caused a significant reduction in the contraction rates in these cardiomyocytes (baseline vs. 0.1 μM Ver: 0.795 ±0.024 vs. 0.245 ±0.012 Hz, n = 5) (Fig. 4Bi–ii). Prolonged treatment at this dose resulted in a complete loss of contracting abilities (data not shown). Contrary to verapamil, isoproterenol caused a significant increase in the beating frequencies (baseline vs. 0.1 μM Iso: 0.657 ± 0.023 vs. 1.68 ± 0.08 Hz, n = 5) (Fig. 4Ci–ii). Interestingly, contractions reverted toward baseline levels following withdrawal of both these drugs (Fig. 4B–C). Furthermore, verapamil also demonstrated a reduction in corrected field potential duration (cFPD) that at 0.1 μM concentration caused a 33% shortening of cFPD (baseline vs. 0.1 μM Ver: 0.302 ±0.053 vs. 0.202 ±0.08 Hz, n = 5). On the other hand, quinidine, a class I antiarrhythmic drug, significantly reduced Na influx by 25% at 1 μM as well as significantly prolonged cFPD (Fig. 4Di–iii). Finally, E-4031, a methanesulfonanilide class III anti-arrhythmic drug, that blocks the hERG-type potassium channel, caused a statistically significant QT prolongation at 100 nM concentration (Fig. 4Ei–ii). Their effects were reversible following removal of the drugs. These studies further validate the presence of functional ion channels suggesting their suitable applications for studying drug effects.

Functional evaluation of cardiomyocytes Majority of our clusters demonstrated beating frequencies in the range of 0.6–0.8 Hz (Fig. 4A). However, intermittently fast (1–1.5 Hz) and slower (0.3–0.5 Hz) contracting clusters too were observed, but their numbers were too low and hence were not included in this study. Following basic characterization of these hiPSC-derived CMs, we next determined if these cardiomyocytes could show functional electrophysiological properties. Beating CM clusters were plated on MEA plates and extracellular field potentials were recorded. We first

Discussion The ability to reprogram differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. This opens an opportunity to offer hope that patient-specific iPSCs could be created to model disease and to generate clinically useful cell types for autologous therapy aimed at repairing deficits arising from injury, illness and aging. Yamanaka and colleagues demonstrated induction

68 G.L. Sequiera et al. / Life Sciences 92 (2013) 63–71 Fig. 3. Ontogenic development of hiPSC-CMs. A, relative gene expression based on real time RT-PCR showing various hallmark markers for cardiac differentiation at day 0, 7, 14 and 21. The mean Ct values of duplicate measurements were calculated and subsequently normalized against housekeeping gene (GAPDH) for the same sample. After normalization, the means of triplicate samples from three independent experiments were plotted relative to the day 7 values of each individual marker, except Oct-4 where day 0 was used for normalization. *p b 0.05 compared to day 7 for all markers, except Oct4 where day 0 is compared. Bi–ii, an overview of temporal gene expression during cardiac differentiation is depicted. Quantitative RT-PCR was performed to determine the trend in expression of genes as depicted above. For gene expression, the level of expression at each time point was normalized to maximal level observed, arbitrarily set as 1. The levels presented herein are mean of three independent experiments representing a trend over time and are not absolute expression levels except for Oct4. The left Y-axis (log scale) represents the absolute expression levels of Oct-4 whereas right Y-axis is arbitrarily set for all differentiated markers.

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Table 1 Correlation analysis between mature genes expressed during differentiation in hiPSC-CM.

CAA cTnT MYH7 MLC2v cTnI CACNA1D KCNH2 KCNQ1

CAA

cTnT

MYH7

MLC2v

cTnI

CACNA1D

KCNH2

KCNQ1

– 0.035 n.s. 0.0004 b0.0001 b0.0001 0.0016 0.0017

0.035 – 0.0382 0.0172 0.0222 0.0391 0.0098 0.0095

n.s. 0.0382 – 0.0456 0.0497 n.s. 0.0395 0.0398

0.0004 0.0172 0.0456 – b0.0001 0.0006 b0.0001 0.0001

0.0001 0.0222 0.0497 b0.0001 – 0.0002 0.0003 0.0004

b0.0001 0.0391 n.s. 0.0006 0.0002 – 0.0022 0.0023

0.0016 0.0098 0.0395 b0.0001 0.0003 0.0022 – b0.0001

0.0017 0.0095 0.0398 0.0001 0.0004 0.0023 b0.0001 –

The table shows gene to gene correlation of inter-relationship between the genes expressed and analyzed in hiPSC-derived cardiomyocytes. The lower the values suggest higher correlation. n.s; not significant. Abbreviations: CAA — cardiac α-actinin; cTnT/I: cardiac troponins T/I; MLC2v: myosin light chain 2, ventricular; MYH7: myosin heavy chain 7; CACNA1D: voltage-gated calcium channel alpha subunit Cav1.3; KCNH2: potassium voltage-gated channel subfamily H member 2; KCNQ1 potassium voltage-gated channel, KQT-like subfamily, member 1.

of pluripotency in somatic cells by enforced expression of four transcriptional factors using retroviral vectors (Takahashi et al., 2007). Although viral based methods have demonstrated high efficiency of reprogramming, the viral integration into the genome presents a formidable obstacle for their eventual therapeutic use (Yu et al., 2009). Recent reports indicate that viral-driven continuous expression of reprogramming genes may influence their differentiation potential or phenotypes of differentiated cell types (Warren et al., 2010). We have previously shown that episomal vectors could be utilized for reprogramming foreskin fibroblasts and these human iPSCs generated could efficiently differentiate into functional cardiomyocytes (Mehta et al., 2011). Nevertheless, reprogramming efficiency with these vectors is reported to be low (3–6 colonies per 106 input cells) (Yu et al., 2009). Despite these low frequencies, sufficient iPSCs can be expected from a reasonable starting cell numbers by this technically straightforward procedure. In the present study, we reprogrammed adult human dermal fibroblast cells using viral-free method, differentiated the generated hiPSCs to cardiac lineage and evaluated their ontogenic development. Furthermore, similarities between hiPSC-CMs and human heart tissue were compared. Our results show that adult somatic cells could be reprogrammed to hiPSC as demonstrated by expression of hallmark pluripotency (Oct4, Sox2 and Nanog) markers that were weakly or not expressed in dermal fibroblasts (Fig. 1). Furthermore, one of the key features of pluripotent stem cells is their ability to differentiate in vitro into three germ layer via embryoid body formation. Our hiPSC differentiated expectedly with expression of markers indicative of ectoderm, mesoderm and endoderm, confirming their pluripotency state (Fig. 1). Furthermore, specific differentiation towards cardiomyogenic lineage was possible for all generated lines. However, it was interesting to note that not all cell lines demonstrated equal cardiomyogenic abilities despite using identical differentiation protocol. While clone AC7 demonstrated highest cardiomyogenic ability (25–35%), clone 2 and 3 had limited propensity toward cardiomyogenic lineage (0–7%). We believe that this may be due to the differentiation propensity or bias of cell lines, a feature previously demonstrated by us and others (Mehta et al., 2010; Osafune et al., 2008) in hESC lines. In order to better understand the developmental pathway of these iPSCs to cardiac lineage, we performed temporal gene expression and our results demonstrated that these hiPSC-CMs recapitulated cardiac developmental program. Our results clearly show that down-regulation of Oct4, the master controller for pluripotency, is the earliest sign of differentiation (Mehta et al., 2008; Niwa et al., 2000). This down-regulation was seen with significant up-regulation of Isl1, a marker for secondary heart field. Further loss of early marker (Isl1) was seen with upregulation of more cardiac committed markers like Nkx2.5, CAA and other sarcomeric proteins by day 14 onwards. This correlates well with

the observation that contracting EBs by day 11–12 suggesting that cardiac machinery has started. However, these contractions were weak and irregular by day 11–12 and required another 5–7 days to show more homogenous contractions. These observations correlated well with gene expression, as most of the mature sarcomeric proteins, troponin and ion channel genes were highly expressed during these days (day 21) resulting in rhythmic contractions. These findings may have important implications for future understanding of cardiac developmental pathways and suggest that current differentiation system and protocol may be open to manipulations to trigger known cardio-mesoderm signaling pathways in an attempt to augment cardiomyocyte yield (Tomescot et al., 2007; Yang et al., 2008). The gene expression results were confirmed at protein levels whereby our hiPSC-CMs demonstrated positive staining for most sarcomeric proteins (Fig. 2). However, it was noteworthy that not all cardiomyocytes show mature cross-striations. While some cardiomyocytes showed striations suggesting maturity, other did not show clear striations. We believe this may be due to lack of mature myofibril structural organization in de novo cardiomyocytes. This could be confirmed by comparing relative gene expression of structural proteins with adult human heart (Fig. 3), which are significantly lower in hiPSCCMs. It is suggested that hiPSC-CMs are phenotypically reminiscent of previously reported 16-week old fetal hearts (Mummery et al., 2003). Our correlation studies demonstrate that there is a significant degree of correlation between hiPSC-CMs and human heart tissue. These observations are in corroboration with recent studies on hESC demonstrating such similarities (Asp et al., 2010). Interestingly, despite having irregular sarcomere structures, these cardiomyocytes expressed appropriate ion channels coupled with downstream signaling pathways that could be modulated by specific cardiac drugs, which further provide functional evidence of their similarities with human heart. We also demonstrated that hiPSC-CMs were able to respond appropriately to drugs known to affect chronotropy. As expected, treatment with isoproterenol increased contraction rates, whereas verapamil arrested them. These results demonstrate the functionality of the derived hiPSC-CMs and support their potential value in drug testing (Flora and Mehta, 2009; Kola and Landis, 2004; Roden, 2004). Generation of iPSC from diseased patients like long QT syndrome (LQTS) resulting in defective QT-CMs, could be of immense value as an in vitro model for drug development and related pharmacogenomics (Itzhaki et al., 2011; Malan et al., 2011; Matsa et al., 2011; Moretti et al., 2010). Conclusions We report characterization and functional verification of viral-free hiPSC-CMs. Our study shows that viral-free hiPSCs are capable of

70 G.L. Sequiera et al. / Life Sciences 92 (2013) 63–71 Fig. 4. Pharmaco-electrophysiological responses of hiPSC-CMs. A, box plot shows the beating frequencies of the two cell lines, AC4 and AC7 (n = 27). B–Ci–ii, extracellular field potential tracing showing alterations in contracting frequencies after treatment with 0.1 μM verapamil (Bi–ii) and 0.1 μM isoproterenol on hiPSC-CMs respectively. Note: while verapamil causes a reduction in contraction, isoproterenol increases contraction rates. Di–iii, single extracellular waveform trace demonstrating the effect of quinidine, class I sodium channel blocker on hiPSC-CMs. Note quinidine causes increase in field potential duration (red arrows) as well as reduction in Na+ current (green arrow). Ei–ii, single extracellular field potential traces showing a dose-dependent prolongation of FPD following E-4031 treatment. In most of the treatments, the waveforms returned towards normalcy following drug withdrawal. Data represent mean ± SEM of three independent experiments. *P b 0.05 as compared to baseline. Abbreviations: B — baseline, V — verapamil, I — isoproterenol, Q — quinidine, E — E4031, W — washout.

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differentiating into cardiomyocytes and recapitulating cardiac developmental process with well-developed electrical properties. Although these cardiomyocytes may be immature, they show a high degree of correlation with the adult human heart. This renewable source of cardiomyocytes may serve as an important in vitro tool for drug screening. Conflict of interest statement None.

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